The surveillance programme for Aphanomyces astaci in Norway 2019

(1)

The surveillance programme for

Aphanomyces astaci in Norway 2019

(2)

The surveillance programme for Aphanomyces astaci in Norway 2019

Content

Summary ...2

Introduction ...3

Aims ...4

Materials and methods ...5

Work plan ...5

Surveillance sites ...5

eDNA monitoring ...7

Results and Discussion ...7

eDNA monitoring in the Halden watercourse ...7

eDNA monitoring in the Mosse watercourse ...8

eDNA monitoring in the Glomma watercourse ...8

eDNA monitoring in Eidskog municipality ...8

Conclusion ... 12

Acknowledgements ... 12

References ... 13

Appendix ... 14

Authors

David Strand¹, Johannes Rusch¹, Stein Ivar Johnsen², Attila Tarpai¹, Trude Vrålstad¹

1 Norwegian Veterinary Institute

2 Norwegian Institute for Nature Research

ISSN 1894-5678

© Norwegian Veterinary Institute 2020

Commissioned by

Norwegian Food Safety Authority

Design Cover: Reine Linjer

Photos front page: Johannes Rusch, David Strand

& Trude Vrålstad

(3)

Summary

In this surveillance programme, environmental DNA (eDNA) monitoring of the water was used as an alternative method to the traditional cage experiments with live noble crayfish. Here, DNA from spores of Aphanomyces astaci were detected directly from water filtrates. The presence/absence of eDNA from noble crayfish (Astacus astacus) and signal crayfish (Pacifastacus leniusculus) was also determined to supplement the results and to gain the possibility to evaluate the habitat status in more detail, and as a part of the collaboration and coordination with the national surveillance programme for noble crayfish.

The main geographic focus of this surveillance programme was on the Halden watercourse and neighbouring risk areas. Other covered geographic areas include the Mosse watercourse, Glomma watercourse, and selected areas in the Eidskog municipality including the Buåa watercourse, the Vrangselva watercourse, and River Finnsrudelva.

In total, 50, 43, 20 and 32 water samples were collected from selected sites in the Halden-, Mosse-, Glomma watercourse regions and in the Eidskog region, respectively. Locations for sampling water were strategically selected and focused on both control zones and the risk areas adjacent to crayfish plague control zones. The presence/absence of the three target species was determined simultaneously through screening with species-specific qPCR assays.

In 2019, eDNA of A. astaci was detected in the lower part of the River Hølandselva in the Halden watercourse while no spread of the pathogen was observed in any of the other monitored areas.

• In the control zone of the Halden watercourse, A. astaci eDNA was detected in the southern part of Lake Rødenessjøen. Here, the known presence of signal crayfish was confirmed by eDNA detection. A.

astaci was also detected in the lower part of the River Hølandselva within the control zone, indicating an active outbreak or infection reservoir in Hølandselva. All water samples in the risk area of the Halden watercourse region were negative for A. astaci and signal crayfish, while most samples were positive for noble crayfish eDNA.

• In the Mosse watercourse, no eDNA of A. astaci or signal crayfish was detected, while noble crayfish eDNA was detected upstream of Lake Langen.

• In the Glomma watercourse, no sign of crayfish plague or signal crayfish was found, while two samples were positive for noble crayfish eDNA.

• In Eidskog municipality, all samples were negative for both signal crayfish and A. astaci, while several were positive for noble crayfish eDNA in River Vrangselva, River Finnsrudelva and River Buåa.

(4)

Introduction

The oomycete Aphanomyces astaci, the causative agent of the crayfish plague, is a lethal pathogen to native European freshwater crayfish (1-3). It is carried and transmitted by North American freshwater crayfish, which act as healthy carriers of the pathogen. A. astaci reproduces and spreads with swimming zoospores, the infective stage of the pathogen. It was accidentally introduced to Europe ~160 years ago and resulted in mass-mortalities of freshwater crayfish all over Europe. It was later re-introduced through many independent introductions of alien North American carrier crayfish (3), particular in signal crayfish.

Crayfish plague is a list 3 disease in Norway, according to the “Regulation on animal health requirements for aquaculture animals and products thereof, prevention and control of infectious diseases in aquatic animals” FOR 2008-06-17-819.

Since 1971, seven water systems in Norway have been affected by crayfish plague outbreaks one or more times (4-5). These include the Vrangselva watercourse and River Veksa (1971), the Glomma watercourse (1987 and 2003), Lake Store Le (1989), the Halden watercourse (1989, 2005 and 2014), River Lysakerelva (1998), Buåa watercourse (2010), and Mosse watercourse (2016). In 2016, crayfish plague was confirmed in noble crayfish inhabiting the watercourse Vrangselva and River Billa shared by Norway and Sweden (which is called River Finnsrudelva on the Norwegian side), but the infection has not yet reached the Norwegian side. In addition, four more localities have been (or still are) subject to crayfish plague regulations due to illegally introduced and confirmed A. astaci positive signal crayfish (4). These include Dammane (Vestfold og Telemark), Ostøya (Viken), Fjelna-vassdraget (Trøndelag) and Kvesjøen (Trøndelag), where signal crayfish were discovered in 2006, 2009, 2011 and 2013, respectively (4-6). At two of these locations (Dammane and Ostøya), signal crayfish have been eradicated and the areas were recently declared disease free after several years of surveillance (4).

The focus areas of the 2019 surveillance programme for crayfish plague cover the

• Halden watercourse (under regulation FOR-2015-05-26-592)

• Mosse watercourse (under regulation FOR-2016-12-13-1523)

• Glomma watercourse (under regulation FOR-2005-06-20-652)

• Eidskog municipality, including Buåa watercourse, Vrangselva watercourse and River Finnsrudelva (under regulation FOR-2016-08-17-972)

The Halden watercourse was first struck by crayfish plague in 1989, re-stocked with noble crayfish in the 1990s and the population successfully recovered until the crayfish plague returned in 2005 (7). Immediate closure of the Ørje locks prevented upstream spread. Illegally introduced A. astaci positive signal crayfish were found in Lake Øymarksjøen in 2008 (8), leading to the permanent closure of the locks. This

prevented further spread, until illegally introduced signal crayfish were found upstream of the locks in 2014. The re-established noble crayfish population in Lake Rødenessjøen was lost during the following plague outbreak (9). In this period, the TARGET project (NRC- 243907) compared cage-based surveillance with environmental DNA (eDNA) monitoring as described in Strand et al. (10). The infection front was followed through analysis of water, and eDNA of A. astaci was sometimes detected in the water samples prior to crayfish mortalities in the cages. Noble crayfish and signal crayfish eDNA were also detected in the locations where the crayfish are known to occur (9).

The Mosse watercourse was struck by crayfish plague in 2016 (11). When the crayfish season started in August, the Norwegian Food Safety Authority (NFSA) received reports regarding possible absence of noble crayfish from Lake Mjærvann and River Hobølelva. No dead crayfish could be found, but eDNA-analyses of water from the small River Tangenelva upstream of Lake Mjærvann (Enebakk) conducted at the Norwegian Veterinary Institute (NVI) confirmed high levels of A. astaci eDNA, corresponding to an outbreak situation (10). The NFSA established zone regulations and initiated surveillance with cages in infected areas. In the cage upstream of the lower dam in the pond Steinkistedammen, the spread of crayfish plague was

detected in December 2016, while the cage placed in Lake Våg was not affected in 2016 (12). No A. astaci eDNA was detected in the Mosse watercource in 2017, but there was a significant drop in eDNA detection

(5)

of noble crayfish from June to August in Lake Våg (12). A dead crayfish found in Lake Langen in 2018 where diagnosed with crayfish plague, confirming the upstream spread of crayfish plague in the watercourse (13).

The Glomma watercourse was struck by crayfish plague in July 1987, from Kirkenær in Solør and further downstream including Lake Vingersjøen and Lake Storsjøen/River Oppstadåa (4). Environment authorities and landowners cooperated to re-establish crayfish in the river system, but the plague struck again in 2003. Cage experiments combined with crayfish plague diagnostics confirmed active crayfish plague in the system from 2005 until 2015 (4-7). The last detection was in the tributary Opstadåa in 2015. No traces of A. astaci eDNA has been detected in the Glomma watercourse, the outlet of Lake Vingersjøen or

Oppstadåa in the period 2016-2018 (6, 12-13), while noble crayfish eDNA was detected in River Oppstadåa in 2016 (6).

The Buåa system was struck by crayfish plague in 2010 caused by the presence of signal crayfish on the Swedish side of the river. A barrier built to prevent the spread of signal crayfish did not stop the infection from spreading, but hopefully stopped the signal crayfish (4). Cage experiments were conducted in the area until 2016 without revealing any active infection source (6). eDNA analysis of samples for Buåa tested negative for A. astaci and signal crayfish in 2017 and 2018 (12-13).

The rivers Vrangselva and Finnsrudelva/Billa in Eidskog municipality, that flow across the border into Sweden were struck by crayfish plague on the Swedish side of the border in 2016. No sign of crayfish plague has been detected on the Norwegian side of the border in either of these two watercourses in 2016 to 2018 (12-13). However, the crayfish plague has been active and slowly migrating upstream in River Finnsrudelva/Billa on the Swedish side of the border in 2017 and 2018.

The surveillance programme for A. astaci is commissioned by NFSA and conducted by NVI. Until 2015, surveillance of crayfish plague relied on cage experiments with live noble crayfish. In 2016, the A. astaci surveillance programme combined the classical cage experiments with eDNA monitoring (6). Here, the eDNA monitoring of A. astaci worked as intended, and in combination with the complementary eDNA targets noble- and signal crayfish, it was possible to produce a snapshot of the relevant habitat status.

Decapods including Noble crayfish are covered by the Norwegian animal welfare act (LOV-2009-06-19-97).

When an alternative method is developed, the use of live animals for disease surveillance should be reduced. Within the cage experiments, the crayfish mortality was 24 % despite the fact that no crayfish plague was detected in any of the ten cages. Furthermore, another 34 % of the crayfish escaped, probably as a result of human interference (vandalism) (6). Based on an overall assessment taking crayfish welfare and cost-benefit into account, the cage experiments were excluded from the surveillance programme in 2017. The programme was co-funded by the research project TARGET in the period 2016-2017. From 2018, a formal collaboration was initiated with the National surveillance programme for noble crayfish (Astacus astacus), commissioned by the Norwegian Environment Agency (NEA) and coordinated by the Norwegian Institute of Nature Research (NINA). This involves joint field work and joint exploitation of water samples and molecular results in overlapping surveillance areas. These synergies enable analyses of a slightly larger sample size than the NFSA-programme alone would allow.

Aims

This surveillance programme aims to

• Monitor the infection pressure and spread of the crayfish plague pathogen A. astaci in zone regulated areas as a result of earlier detection of disease (referred to as control zones).

• Substantiate disease free waterbodies in neighbouring areas of the control zones (= risk areas).

• Alert the authorities of any eventual spread of the disease from control zone to risk areas.

• Continue to evaluate eDNA as a monitoring tool for A. astaci - alone and in combination with complementary eDNA targets including both the carrier and susceptible crayfish host species.

(6)

Materials and methods

Work plan

The surveillance programme is based on eDNA monitoring of water, where DNA from spores of A. astaci are detected directly from water filtrates. To complement information on the habitat status, eDNA from the native and susceptible noble crayfish A. astacus and the alien carrier signal crayfish P. leniusculus is monitored within the same water samples. The logistics and analyses are conducted in collaboration with the national surveillance of noble crayfish, funded by NEA, and coordinated by NINA (Figure 1).

Surveillance sites

The main areas for surveillance include the Halden watercourse and surrounding areas, the Mosse watercourse, the Glomma watercourse, and Eidskog municipality including the Vrangselva watercourse, Buåa watercourse and River Finnsrudelva. Plotted locations for water sampling, in total 35 sites, as well as the crayfish plague zones, are displayed in Figure 2. Supplementary details are summarised in Appendix 1 (Table S2-S5).

Halden watercourse: The control zone was monitored at a total of 5 sites from Lake Fossersjøen to the outlet of Lake Rødenessjøen (Ørje locks). Live noble crayfish were still expected to be present within the control zone in the upper parts of the system, awaiting the outbreak. Crayfish localities adjoining the control zone or in close geographical proximity are vulnerable to further spread and referred to as "risk zones" (Table S2, Appendix 1). In total, 7 sites were monitored in the risk zone.

Mosse watercourse: The control zone was monitored from Lake Sværsvann and Lake Binningsvann and downstream to River Hobølelva, in total 10 sites (Table S3, Appendix 1).

Glomma watercourse: The control zone comprises the main passageway downstream Braskereidfoss in Våler. Five sites within the control zone was monitored. (Table S4, Appendix 1).

Eidskog: The control zone (defined by the municipality border) was monitored in the Vrangselva

watercourse (4 sites), Buåa watercourse (2 sites) and River Finnsrudelva (2 sites) (Table S5, Appendix 1).

Figure 1. Work plan: The Norwegian Veterinary Institute (NVI) coordinates the project, and organises the eDNA water sampling and qPCR screenings in collaboration with the national surveillance of noble crayfish (Funded by the Norwegian Environment Agency (NEA).

(7)

Figure 2. Surveillance sites in Eastern Norway 2019. Water samples (circles) were collected in June and September. Regulated areas (crayfish plague control zones) are marked in red. Note: For Glomma, the control zone is an approximation.

(8)

eDNA monitoring

The water samples were collected in June and September 2019. From each site, two samples of ~5 L were filtered through sterile glass fibre filters on-site (10). Ideally, 5 L was filtered per filter sample, but due to high turbidity or clay particles, the total filtered volume was sometimes lower. In some of these cases extra samples were included to partly compensate for the reduced water volume. This explains the increased number of samples at some sites (Table S2-S5) compared to the agreed number of samples (Table S1).

The filters were transferred with a clean forceps to a ziplock bag containing silica-gel beads, which dry the filter to preserve biological material and DNA.

The water samples were screened by qPCR for three DNA targets: the species-specific qPCR assay for A.

astaci (10, 14) and two crayfish species specific qPCR assays for noble crayfish and signal crayfish developed by Agersnap et al. (15). Figure 3 presents an overview of the eDNA monitoring procedure.

Figure 3. Water samples of ~5 L each were filtered on-site through glass fibre filters using a portable peristaltic pump (Masterflex E/S portable sampler). Each filter was carefully transferred to a ziplock bag containing silica-gel beads for preservation until transportation to the laboratory. DNA was isolated with a large volume extraction procedure and presence/absence of eDNA from all target organisms was analysed using qPCR. Figure modified from Vrålstad et al (6).

Results and Discussion

eDNA monitoring in the Halden watercourse

In the Halden watercourse region, 50 water samples representing a total of ~210 L water were analysed.

In the control zone, A. astaci eDNA was detected in two water samples (in June) at the Southern part of lake Rødenessjøen (Figure 4, Table S2) where signal crayfish were confirmed to be present by positive eDNA results in a total of three water samples (two in June, one in September; Figure 4, Table S2). eDNA from A. astaci was also detected in one sample at the outlet of River Hølandselva in September, where the plague was last detected in 2016. It is uncertain if the detection at the outlet of River Hølandselva represents a new introduction of crayfish plague into the river or if it has survived undetected in the river system for several years.

No sign of crayfish plague was observed at any other station upstream the outlet of Hølandselva in June or September, indicating that the outbreak is restricted to the lower parts of River Hølandselva. These results were supported by positive detections of noble crayfish eDNA in samples from River Hølandselva and upstream (in the control zone), indicating the presence of live noble crayfish inhabiting the northern

(9)

part of the Halden watercourse control zone. In total, eDNA of noble crayfish was detected in 5 water samples from River Hølandselva and upstream (in the control zone).

In general, all water samples from the risk area surrounding the Halden watercourse were negative for A.

astaci and signal crayfish eDNA, while most samples were positive for noble crayfish eDNA. In total, 18 water samples were positive for noble crayfish eDNA (Figure 4, Table S2). The combined absence of A.

astaci eDNA and presence of noble crayfish eDNA suggests that there has been no further spread of the disease in the surveillance period, and that there are live noble crayfish at the monitored sites. This was supported by CPUE (catch per unit effort) data from the national surveillance programme for noble crayfish 2019 (16), where live noble crayfish were documented in Lake Hemnessjøen at a density of 4.96 crayfish per trap-night (CPUE).

eDNA monitoring in the Mosse watercourse

In the Mosse watercourse, 43 water samples representing a total of ~165 L water were analysed. None of the analysed samples showed any sign of A. astaci or signal crayfish eDNA (Figure 5, Table S3). eDNA from noble cayfish was detected in six samples (four in June and two in September) at two stations upstream Lake Langen. This suggests that crayfish plague has not spread upstream from Lake Langen where crayfish plague was confirmed in 2018 after positive detection on one dead crayfish found at Kilevika.

eDNA monitoring in the Glomma watercourse

In the Glomma watercourse, 20 water samples representing a total of ~93 L water were analysed. No sign of A. astaci or signal crayfish was found through eDNA analysis (Figure 6, Table S4). Three samples were positive for noble crayfish eDNA (in June) at two stations in Glomma. The results cannot verify any active A. astaci infection or infection source from the monitored sites in the Glomma.

eDNA monitoring in Eidskog municipality

In the Eidskog municipality, 32 water samples representing a total of ~148 L water were analysed. None of the analysed samples showed any sign of A. astaci or signal crayfish (Figure 6, Table S5). In the Vrangselva watercourse, six samples from Åbogen to Skotterud were positive for noble crayfish eDNA (four in June, two in September), suggesting that the river stretch is still inhabited by live noble crayfish. In River Finnsrudelva, four samples were positive for noble crayfish eDNA (in September).

In the Buåa watercourse, two samples were positive for noble crayfish eDNA (in June). No sign of A. astaci or signal crayfish was found through eDNA analysis (Figure 6, Table S5). The Buåa watercourse has been monitored by cages for more than five years (6) and by eDNA alone for another three years. Lack of crayfish plague detection could indicate disease free status. However, a new crayfish plague regulation from August 2016 covers the whole Eidskog municipality (FOR-2016-08-17-972), and replaces the old regulation for the Buåa watercourse. Thus, as long as the Eidskog region is covered by one regulation, no conclusion can yet be drawn regarding disease freedom in the Buåa watercourse.

(10)

Figure 4. Overview map of the surveyed part of the Halden watercourse region in 2019, starting from the Ørje locks (black arrow) in the south where signal crayfish is present. The control area is indicated by red colour on involved lakes and rivers, and ends at Fosserdam (red arrows), which is an artificial barrier for further spread. The pie chart indicates presence (colour) or absence (white) of A. astaci (red), signal crayfish (P. leniusculus; yellow), and noble crayfish (A. astacus; green). Presence is listed if at least one of the tested water samples yielded a positive eDNA result. Positive A. astaci samples were detected close to Ørje locks together with P. leniusculus eDNA and at the outlet from River Hølandselva. Only eDNA of noble crayfish was detected in the water upstream the outlet of River Hølandselva. The same was observed in the risk area, suggesting no spread of A. astaci in the monitoring period.

(11)

Figure 5. Overview map of the surveyed part of the Mosse watercourse. The control area, which was extended in 2018 due to crayfish plague in Lake Langen, is represented by red colour. The pie chart indicates presence (colour) or absence (white) of A.

astaci (red), signal crayfish (P. leniusculus; yellow), and noble crayfish (A. astacus; green). Presence is listed if at least one of the tested water samples yielded a positive eDNA result. No eDNA of A. astaci and signal crayfish was detected.

(12)

Figure 6. Overview map of the Glomma watercourse region and Eidskog municipality. Regulated areas (crayfish plague control zones) are marked in red. For each location site, the pie chart indicates presence (colour) or absence (white) of A. astaci (red), signal crayfish (P. leniusculus; yellow), and noble crayfish (A. astacus; green). Presence is listed if at least one of the tested water samples yielded a positive eDNA result. Only eDNA from noble crayfish were detected in the Glomma watercourse. In the Vrangselva

watercourse, no eDNA of A. astaci or signal crayfish was detected, while eDNA of noble crayfish was detected at Åbogen and Magnor.

Also, in the River Finnsrudelva, eDNA of noble crayfish was detected but without signs of A. astaci or signal crayfish. Only eDNA from noble crayfish were detected in the Buåa watercourse.

(13)

Conclusion

In the Halden watercourse, combined eDNA monitoring of A. astaci, noble crayfish and signal crayfish confirmed that signal crayfish present in Lake Rødenessjøen emit detectable, but low concentrations of A.

astaci to the water. The detection of A. astaci in the lower part of River Hølandselva indicates that there is an active infection front in the river. Since there was no detection of A. astaci at River Hølandselva in 2017 and 2018 (12-13), it is uncertain if this represents a new introduction or if the plague has gone undetected until now. One likely scenario is that the crayfish plague has survived and slowly spread in parts of the river with very low crayfish densities in 2017 and 2018, and has reached a part of the river with higher densities of crayfish in 2019. There was no sign of A. astaci in the northern part of River Hølandselva or in any of the stations in the neighbouring risk areas indicating that the outbreak is limited to the lower part of River Hølandselva. This is also supported with positive samples for noble crayfish eDNA at all stations upstream.

No A. astaci positive eDNA samples were observed in the Mosse watercourse in this surveillance

programme in 2019 at the surveyed sites in June and September. While the crayfish plague reached Lake Langen in 2018 (13), detection of noble crayfish eDNA upstream the lake indicates no further spread.

In the Glomma watercourse, no A. astaci or signal crayfish eDNA was detected. The status is still highly uncertain, given many years of recurrent crayfish plague detection in cage experiments. However, the results indicate at least that our sampling effort was not sufficient to reveal an eventual infection source in the watercourse. Positive eDNA results for noble crayfish was found in 2016 and 2019, but not in 2017 and 2018 (12-13).

We found no sign of A. astaci in any of the monitored sites in Eidskog municipality. Similar to the results of 2017 and 2018 (12-13), noble crayfish eDNA was detected at several of the monitored sites in the Vrangselva watercourse and River Finnsrudelva. This supports the view that the crayfish plague has still not yet entered the Norwegian side of these river systems and suggests the presence of live noble crayfish in both systems. However, the crayfish plague was active in River Finnsrudelva at the border in June 2018 (17-18). Noble crayfish DNA was also detected in the Buåa watercourse.

Acknowledgements

Both the surveillance programme for Aphanomyces astaci in Norway and the national surveillance programme of noble crayfish benefit from the coordinated and simultaneous monitoring of the three target species (crayfish plague, red-listed noble crayfish and black-listed signal crayfish). We thank the Norwegian Food Safety Authority (NFSA) for accepting the idea of a joint collaborative project between these surveillance programmes. The surveillance programme for A. astaci is funded by NFSA, and the national surveillance programme for noble crayfish is funded by the Norwegian Environment Agency (NEA).

(14)

References

1. Alderman DJ, Polglase JL, Frayling M. 1987. Aphanomyces astaci pathogenicity under laboratory and field conditions. Journal of Fish Diseases 10: 385-393.

2. Holdich DM, Reynolds JD, Souty-Grosset C, Sibley PJ. 2009. A review of the ever increasing threat to European crayfish from non-indigenous crayfish species. Knowledge and Management of Aquatic Ecosystems 394-395, 11.

3. Söderhäll K, Cerenius L. 1999. The crayfish plague fungus: History and recent advances. Freshwater Crayfish 12:

11-35.

4. Johnsen SI, Vrålstad T. 2017. Edelkreps (Astacus astacus) - Naturfaglig utredning og forslag til samordning av overvåkingsprogrammene for edelkreps og krepsepest- NINA Rapport 1339. 39 s.

5. Vrålstad T, Strand DA, Grandjean F, Kvellestad A, Håstein T, Knutsen AK, Taugbøl T, Skaar I. 2014. Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history. Veterinary Microbiology 173: 66-75.

6. Vrålstad T, Strand D, Rusch J, Toverud Ø, Johnsen SI, Tarpai A, Møller PR, Gjevre AG. 2017. The surveillance programme for Aphanomyces astaci in Norway 2016. Annual Report 2016. ISSN 1894-5678. Norwegian Veterinary Institute, 16 pp. Available at: https://www.vetinst.no/en/surveillance-programmes/crayfish-plague-

aphanomyces-astaci

7. Vrålstad T, Håstein T, Taugbøl T, Lillehaug A. 2006. Krepsepest - smitteforshold i norske vassdrag og forebyggende tiltak mot videre spredning av krepsepest, 1-25. Veterinærinstituttet rapportserie 6-2006.

8. Vrålstad T, Johnsen SI, Fristad RF, Edsman L, Strand DA. 2011. Potent infection reservoir of crayfish plague now permanently established in Norway. Diseases of Aquatic Organisms 97: 75-83

9. Strand DA, Johnsen SI, Rusch JC, Agersnap S, Larsen WB, Knudsen SW, Møller PR, Vrålstad T. 2019. Monitoring a Norwegian freshwater crayfish tragedy - eDNA snapshots of invasion, infection and extinction. Journal of Applied Ecology 56:1661-1673

10. Strand DA, Jussila J, Johnsen SI, Viljamaa-Dirks S, Edsman L, Wiik-Nielsen J, Viljugrein H, Engdahl F, Vralstad T.

2014. Detection of crayfish plague spores in large freshwater systems. Journal of Applied Ecology 51: 544-553.

11. Krepsepesten har spredt seg i Mossevassdraget. 05.12.2016. Veterinærinstituttet.no:

https://www.vetinst.no/nyheter/krepsepesten-har-spredt-seg-i-mossevassdraget

12. Vrålstad T, Rusch J, Johnsen SI, Tarpai A, Strand D. 2018. The surveillance programme for Aphanomyces astaci in Norway 2017. Annual Report 2017. ISSN 1894-5678. Norwegian Veterinary Institute, 16pp. Available at:

https://www.vetinst.no/en/surveillance-programmes/crayfish-plague-aphanomyces-astaci

13. Strand D, Rusch J, Johnsen SI, Tarpai A, Vrålstad T. 2019. The surveillance programme for Aphanomyces astaci in Norway 2018. Annual Report 2018. ISSN 1894-5678, Norwegian Veterinary Institute, 16pp. Available at:

https://www.vetinst.no/en/surveillance-programmes/crayfish-plague-aphanomyces-astaci

14. Vrålstad T, Knutsen AK, Tengs T, Holst-Jensen A. 2009. A quantitative TaqMan^®MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci. Veterinary Microbiology 137: 146-155.

15. Agersnap S, Larsen WB, Knudsen WS, Strand DA, Thomsen PF, Hesselsøe M, Mortensen PB, Vrålstad T, Møller PR.

2017. Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples.

Plos One 12: e0179261. DOI: 10.1371/journal.pone.0179261

16. Johnsen SI, Strand DA, Rusch J, Vrålstad T. 2019. Nasjonal overvåking av edelkreps og spredning av signalkreps - presentasjon av overvåkingsdata og bestandsstatus – oppdatert 2019. NINA Rapport 1761. 106 s. + vedlegg.

17. Krepsepesten har nådd norskegrensen i Billa. 21.12.2017. Veterinærinstituttet.no:

https://www.vetinst.no/nyheter/krepsepesten-har-nadd-norskegrensen-i-billa

18. Ny mobilteknologi kan påvise krepsepestsmitte direkte i felt. 25.06.2018. Veterinærinstituttet.no https://www.vetinst.no/nyheter/ny-mobilteknologi-kan-pavise-krepsepestsmitte-direkte-i-felt

(15)

Appendix

Supplementary information to the report “The surveillance programme for Aphanomyces astaci in Norway 2019” - Tables S1 – S5.

Table S1. Agreed areas and locations of the “NOK A. astaci 2019” program. We reserve the right to change and a reallocation of sample localities if new circumstances arise.

Location Watercourse¹/

municipality, county² Location infection status # water samples (site X samples X visits) Halden

watercourse Total samples 48

Rødenessjøen HW/Marker, Ø Control zone 8 (2 x 2 x 2)

Hølandselva HW/Aurskog-Høland, A Control zone 8 (2 x 2 x 2)

Fossersjøen HW/Aurskog-Høland, A Control zone, outbreak expected 4 (1 x 2 x 2) Fosserdam overside HW/Aurskog-Høland, A Risk zone/control zone border 4 (1 x 2 x 2)

Bjørkelangen HW/Aurskog-Høland, A Risk zone 4 (1 x 2 x 2)

Lierelva HW/Aurskog-Høland, A Risk zone 4 (1 x 2 x 2)

Lundsfoss HW/Aurskog-Høland, A Risk zone 4 (1 x 2 x 2)

Dalstorpsfoss HW/Aurskog-Høland, A Risk zone 4 (1 x 2 x 2)

Hemnessjøen Lake/Aurskog-Høland, A Risk zone 8 (2 x 2 x 2)

Glomma

Storsjøen GW/Nord & Sør Odal, H Control zone 4 (1 x 2 x 2)

Oppstadåa GW/Sør-Odal, H Control zone 8 (2 x 2 x 2)

Vingersnoret GW/ Sør-Odal, H Control zone 4 (1 x 2 x 2)

Vingersjøen GW/ Sør-Odal, H Control zone 4 (1 x 2 x 2)

Eidskog Total samples 32

Buåa BW/Eidskog, H Control zone 8 (2 x 2 x 2)

Finnsrudelva RF/Eidskog, H Control zone 8 (2 x 2 x 2)

Vrangselva VW/Eidskog, H Control zone 16 (4 x 2 x 2)

Mosse

Hobølelva MV/Enebakk, Ø Control zone 4 (1 x 2 x 2)

Mjær MV/Enebakk, Ø Control zone 4 (1 x 2 x 2)

Tangenelva MV/Enebakk, Ø Control zone 4 (1 x 2 x 2)

Våg badeplassen MV/Enebakk, Ø Control zone 4 (1 x 2 x 2)

Langen MV/Enebakk, Ø Control zone 8 (2 x 2 x 2)

Upstream Langen MV/Enebakk, Ø Control zone 16 (4 x 2 x 2)

Total 140

1 HW = Halden watercourse, GW = Glomma watercourse, MW = Mosse-watercourse, BW = Buåa watercourse, RF = River Finnsrudelva, VW = Vrangselva watercourse

2 Ø = Østfold, A = Akershus, H = Hedmark.

(16)

Table S2. Locations for water sampling in the Halden watercourse area with corresponding location and sample information. eDNA results are listed for crayfish plague, noble crayfish and signal crayfish.

Location¹ Location details Water

samples²

# eDNA positive samples³ June September ID S¹ GPS coordinates # L CP NC SC CP NC SC

Lierelva HA1 R 59°53’8"N 11°34’29"E 4 18 0 2 0 0 1 0

Bjørkelangen HA2 R 59°50’55"N 11°31’5"E 4 20 0 1 0 0 1 0

Fosserdam HA3 R 59°49’17"N 11°29’27"E 4 20 0 1 0 0 0 0

Fossersjøen HA4 C 59°48’58"N 11°29’32"E 2 10 0 1 0 NA NA NA

Lundsfoss HA5 R 59°42’7"N 11°32’14"E 4 20 0 2 0 0 2 0

Hemnessjøen pier HA6 R 59°41’47"N 11°25’7"E 5 21 0 0 0 0 1 0 Hemnessjøen outlet HA7 R 59°43’31"N 11°25’11"E 5 15 0 1 0 0 1 0 Daltorpsfoss HA8 R 59°43’13"N 11°28’49"E 6 13.3 0 2 0 0 3 0 Hølandselva north HA9 C 59°46’7"N 11°29’8"E 4 19.5 0 0 0 0 1 0 Hølandselva outlet HA10 C 59°40’30"N 11°31’50"E 4 17 0 1 0 1 2 0 Rødnessjøen Kroksund HA11 C 59°37’6"N 11°35’5"E 4 16.5 0 0 0 0 0 0 Rødenessjøen Ysterud HA12 C 59°29’17"N 11°38’23"E 4 20 2 0 2 0 0 1

Total 50 210.3 2 11 2 1 12 1

1 C = Crayfish plague control zone, R = risk area

2 # = Total number of water samples (June & September summarized), L = total water volume summarized for all samples

3 Number of samples in June and September with positive detection of eDNA from crayfish plague (CP), noble crayfish (NC), and signal crayfish (SC).

NA = Not available.

Table S3. Locations for water sampling in Mosse-watercourse area with corresponding location and sample information. eDNA results are listed for crayfish plague, noble crayfish and signal crayfish.

Location Location details Water

samples² # eDNA positive samples³ June September ID S¹ GPS coordinates # L ^CP NC SC CP NC SC Bindingsvann, outlet MO11 C 59°47'22.1"N 10°57'17.6"E 4 16 0 0 0 0 0 0 Tangentjern, inlet,

bridge on Brusagaveien MO12 C 59°47'18.2"N 10°54'02.9"E 4 21 0 2 0 0 1 0 Sværsvann MO8 C 59°49'03.2"N 10°53'25.3"E 4 20 0 0 0 0 0 0 Tangentjern, inlet,

bridge on Hareveien MO10 C 59°47'25.7"N 10°53'27.5"E 4 12.3 0 0 0 0 0 0 Langen, inlet, bridge

on Bru-fjellv. MO9 C 59°46'44.7"N 10°54'38.6"E 4 15 0 2 0 0 1 0 Langen, bridge on

Skiveien MO1 C 59°43'33.3"N 11°00'12.1"E 5 13 0 0 0 0 0 0

Våg MO2 C 59°44'10.2"N 11°01'14.7"E 5 13.6 0 0 0 0 0 0

Tangenelva, bridge on

Tomterveien MO5 C 59°43'19.9"N 11°03'18.9"E 4 17 0 0 0 0 0 0 Mjær, outlet MO6 C 59°41'10.2"N 11°02'27.6"E 5 16.6 0 0 0 0 0 0 Hobølelva, Elvestad MO7 C 59°37'26.5"N 10°57'09.2"E 4 20 0 0 0 0 0 0

Total 43 164.5 0 4 0 0 2 0

1 C = Crayfish plague control zone, R = risk area

(17)

Table S4. Locations for water sampling in the Glomma region with corresponding location and sample information.

eDNA results are listed for crayfish plague, noble crayfish and signal crayfish.

samples² # eDNA positive samples³ June September ID S¹ GPS coordinates # L CP NC SC CP NC SC Vingersnoret GL1 C 60°11’36.3"N 12°01’54.5"E 4 16 0 0 0 0 0 0 North of Vingersnoret GL2 C 60°11’39.7"N 12°01’41.2"E 4 20 0 2 0 0 0 0 Storsj. Ringåsvn. pier GL5 C 60°20'18.4"N 11°38'36.5"E 4 18 0 0 0 0 0 0 Oppstadåa south GL9 C 60°16’40.3"N 11°39’06.9"E 4 18 0 0 0 0 0 0 Glomma, Skarnes GL10 C 60°15'20.8"N 11°40'49.4"E 4 20.5 0 1 0 0 0 0

Total 20 92.5 0 3 0 0 0 0

1 C = Crayfish plague control zone

Table S5. Locations for water sampling in the Eidskog region with corresponding location and sample information.

eDNA results are listed for crayfish plague, noble crayfish and signal crayfish.

samples² # eDNA positive samples³ June September ID S¹ GPS coordinates # L ^CP NC SC CP NC SC Vrangselva, Åbogen VR1 C 60°06'43.6"N 12°07'01.0"E 4 21 0 0 0 0 2 0 Søndre Åklangen,

Badeplass VR2 C 60°03'12.8"N 12°08'20.8"E 4 18.5 0 2 0 0 0 0 Vrangselva, Skotterud VR3 C 59°58'53.8"N 12°07'19.1"E 4 18 0 2 0 0 0 0 Vrangselva, Magnor

bad VR4 C 59°57'02.7"N 12°11'58.8"E 4 16 0 0 0 0 0 0 Finnsrudelva,

Finnsrudvegen FR1 C 59°59'50.7"N 12°19'05.4"E 4 20 0 0 0 0 2 0 Finnsrudelva,

Billavegen FR2 C 59°58'44.9"N 12°20'14.2"E 4 20 0 0 0 0 2 0 Buåa, Eidskog BU1 C 59°55'31.1"N 11°59'37.0"E 4 17 0 1 0 0 0 0 Buåa, Riksgrense BU2 C 59°53'56.4"N 11°59'12.0"E 4 17.5 0 1 0 0 0 0

Total 32 148 0 6 0 0 6 0

1 C = Crayfish plague control zone

(18)

Trondheim

[email protected] Sandnes

[email protected] Bergen

[email protected] Harstad

[email protected] Tromsø [email protected] [email protected]Oslo

www.vetinst.no The Norwegian Veterinary Institute is a national research institute that operates in the fields of animal and fish health, food safety and feed hygiene; its primary task is to provide the authorities with independently generated knowledge.

Emergency preparedness, diagnostic services, monitoring, reference functions, consulting, and risk assessments are all important areas of activity. Our products and services include research results and reports, analyses and diagnoses, studies and advice.

The Norwegian Veterinary Institute’s central laboratory and administration lie in Oslo, and we operate regional laboratories in Sandnes, Bergen, Trondheim, Harstad and Tromsø.

The Norwegian Veterinary Institute collaborates with a large number of national and international institutions.

– for integrated health

Fish health Animal health Food safety

Fish health Animal health Food safety